In recent decades, drug-protein interactions have been widely studied and several methods of analysis of these phenomena have been developed and improved. These can be classified into separation, physical, chromatographic and electrophoretic methods. This review depicts the assumptions and mechanisms of methods from each group, details their strengths and weaknesses, and presents examples of their usage from the literature. Equilibrium dialysis, ultrafiltration, Hummel-Dreyer method or high performance affinity chromatography are given as representative examples, but this issue is far more expanded. Nowadays, increasing attention is paid to the computational methods and molecular modeling which are convenient tools to estimate protein binding affinity based on the physicochemical properties of compounds. To gain a broader overview, the study also examines the protein binding ability and pharmacotherapy of drugs against a range of substrates such as plasma, skin, tissue and human milk.